One of the key immune regulators is the T helper cell which reacts to antigens presented on HLA class II molecules. This CD4+ cell differentiates in response to antigenic stimulation and becomes a type 1 or type 2 helper (Th1 or Th2) according to the type of cytokines that it secretes. Mosmann and Coffman, Ann. Rev. Immunol. 7:145-173 (1989). A Th1 response leads to the secretion of interleukin-2 (IL-2) and interferon-γ (IFN-γ) which stimulates cell-mediated immune reactions against intracellular pathogens. A Th2 response leads to the secretion of IL-4, IL-5 and IL-10 which stimulates antibody responses to extracellular pathogens. The most interesting component of this system of regulation is that one response inhibits the other through the negative regulatory activities of the cytokines that are produced. Thus, IL-4 and IL-10 can down-regulate Th1 responses while IFN-γ can down-regulate Th2 responses.
The regulatory activity of T helper cells and their differentiation following exposure to antigen is regulated by cytokines as well. IL-12, a disulfide-linked heterodimeric cytokine with a 40 kDa subunit and 35 kDa subunit, exerts a powerful positive regulatory influence on the development of Th1 helper T-cell immune responses. See review by Trinchieri, Blood 84: 4008-4027 (1994). IL-12 also has a powerful synergistic effect in the induction of IFN-γ from both T helpers and natural killer (NK) cells (Eur. Patent Appl. 90123670.3). Secreted IFN-γ then inhibits any Th2 cell proliferation and polarizes the response to favor cell-mediated immunity.
One way of changing the outcome of an immune response would be to administer the appropriate cytokine at the time of antigen stimulation. If IL-4 was the major cytokine present during antigen stimulation, the Th2 response would be enhanced and the Th1 response would be inhibited. In contrast, if IL-12 was the major cytokine present during antigen stimulation, the Th1 response would be enhanced and the Th2 response would be inhibited. However, systemic administration of cytokines is difficult due to their very short circulating half-lives and their deleterious side effects.
A better approach is to target the effect of the cytokine to a cell surface antigen by fusing it to an antibody (or fragment derived therefrom) having specificity and affinity for that antigen. See Gillies, et al., Proc. Natl. Acad. Sci. 89: 1428-1432 (1992); U.S. Pat. No. 5,650,150, the disclosure of which is incorporated herein by reference. Alternatively, the stimulatory cytokine can be linked to a protein antigen via a peptide linkage in the form of a fusion protein. See Hazama, et al., Vaccine 11: 629-636 (1993). However, the complex structure of IL-12 makes it more difficult to express as a fusion protein due to the necessity of expressing exactly the same molar ratio of each subunit in the final product. In fact, IL-12 itself is naturally expressed and secreted as a mixture of p40 homodimer. D'Andrea, et al., J. Exp. Med., 176:1387-1398 (1992).
Therefore, there is a need in the art for methods of producing fusion proteins with heterodimeric cytokines and an antibody or an antigen that maintain the natural heterodimeric structure of the cytokine and secretes the molecules with equimolar ratios of the subunits.